Quinazoline-4-piperidine sulfamides are specific inhibitors of human NPP1 and prevent pathological mineralization of valve interstitial cells.

BACKGROUND AND PURPOSE: Ectonucleotide pyrophosphatase/PDE1 (NPP1) is an ectoenzyme, which plays a role in several disorders including calcific aortic valve disease (CAVD). So far, compounds that have been developed as inhibitors of NPP1 lack potency and specificity. Quinazoline-4-piperidine sulfamides (QPS) have been described as potent inhibitors of NPP1. However, their mode of inhibition as well as their selectivity and capacity to modify biological processes have not been investigated. EXPERIMENTAL APPROACH: In the present series of experiments, we have evaluated the efficacy of two derivatives, QPS1-2, in inhibiting human NPP1, and we have evaluated the effect of the most potent derivative (QPS1) on other ectonucleotidases as well as on the ability of this compound to prevent phosphate-induced mineralization of human primary aortic valve interstitial cells (VICs). KEY RESULTS: The QPS1 derivative is a potent (Ki 59.3 ± 5.4 nM) and selective non-competitive inhibitor of human NPP1. Moreover, QPS1 also significantly inhibited the K121Q NPP1 gene variant (Ki 59.2 ± 14.5 nM), which is prevalent in the general population. QPS1 did not significantly alter the activity of other nucleotide metabolizing ectoenzymes expressed at the cell surface, namely NPP3, NTPDases (1-3), ecto-5'-nucleotidase and ALP. Importantly, QPS1 in the low micromolar range (≤10 µM) prevented phosphate-induced mineralization of VICs and lowered the rise of osteogenic genes as expected for NPP1 inhibition. CONCLUSIONS AND IMPLICATIONS: We have provided evidence that QPS1 is a potent and selective non-competitive inhibitor of NPP1 and that it prevented pathological mineralization in a cellular model.

A novel bio-orthogonal cross-linker for improved protein/protein interaction analysis.

The variety of protein cross-linkers developed in recent years illustrates the current requirement for efficient reagents optimized for mass spectrometry (MS) analysis. To date, the most widely used strategy relies on commercial cross-linkers that bear an isotopically labeled tag and N-hydroxysuccinimid-ester (NHS-ester) moieties. Moreover, an enrichment step using liquid chromatography is usually performed after enzymatic digestion of the cross-linked proteins. Unfortunately, this approach suffers from several limitations. First, it requires large amounts of proteins. Second, NHS-ester cross-linkers are poorly efficient because of their fast hydrolysis in water. Finally, data analysis is complicated because of uneven fragmentation of complex isotopic cross-linked peptide mixtures. We therefore synthesized a new type of trifunctional cross-linker to overrule these limitations. This reagent, named NNP9, comprises a rigid core and bears two activated carbamate moieties and an azido group. NNP9 was used to establish intra- and intermolecular cross-links within creatine kinase, then to map the interaction surfaces between α-Synuclein (α-Syn), the aggregation of which leads to Parkinson's disease, and the molecular chaperone Hsc70. We show that NNP9 cross-linking efficiency is significantly higher than that of NHS-ester commercial cross-linkers. The number of cross-linked peptides identified was increased, and a high quality of MS/MS spectra leading to high sequence coverage was observed. Our data demonstrate the potential of NNP9 for an efficient and straightforward characterization of protein-protein interfaces and illustrate the power of using different cross-linkers to map thoroughly the surface interfaces within protein complexes.

Synthesis, biological evaluation, and in vivo imaging of the first camptothecin-fluorescein conjugate.

The first synthesis and photophysical properties of a fluorecently labeled camptothecin derivative, namely, camptothecin-FI (CPT-FI), an antitumoral agent that targets topoisomerase I, are reported. The preparation of this fluorescent conjugate is based on a highly convergent and flexible approach which enables the rapid chemical modification of the AB ring system of this fragile pentacyclic alkaloid, aimed at introducing an anchoring point to graft the fluorophore. The selection of a fluorescein analogue as the reporter group has enabled us to get the first green-emitting CPT conjugate exhibiting valuable spectral properties and retaining biological properties of native CPT. Indeed, in biological models, i.e., glioma cell lines U87 and/or T98, the kinetics of cell endocytosis, as well as the efficacy of CPT-FI were compared to those of CPT. CPT-FI fluorescence was measured in the cytosolic compartment of T98 glioma cells from 5 min treatment and remained detectable until 48 h. As CPT, CPT-FI drastically inhibited glioma growth and cell cycle but exhibited a reduced affinity as compared to the native CPT. In vivo and ex vivo imaging studies of CPT-FI intratumoraly injected into a model of NIH-3T3 murine tumor xenografts in nude mice, showed accumulation around the injected site area, which is very promising to target tumors and follow biodistribution in vivo.

The first "ready-to-use" benzene-based heterotrifunctional cross-linker for multiple bioconjugation.

The synthesis and applications of the first water-soluble benzene derivative bearing a set of three different and orthogonal bioconjugatable groups (aminooxy, azido and thiol) are described. The combined use of a 5-amino isophthalic acid scaffold and unusual acid-labile protecting groups for temporarily masking aminooxy and thiol moieties has enabled the development of a highly convergent approach towards the synthesis of such a trivalent bioconjugation platform in good yields. The potential utility of this "ready-to-use" cross-linking reagent for creating complex and fragile tri-component (bio)molecular systems was illustrated through (1) the rapid preparation of a three-colour FRET cascade with valuable spectral properties and (2) the luminescent/fluorescent labelling of peptides and peptide-oligonucleotide conjugates. Thus, such (bio)molecular assemblies were readily obtained via a three-step process or in a "one-pot" manner, both involving oxime ligation, thiol-alkylation (S(N)2 or Michael addition) and copper-catalysed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reactions.

New insights into the water-solubilisation of fluorophores by post-synthetic "click" and Sonogashira reactions.

New synthetic methodologies for the efficient chemical conversion of hydrophobic fluorescent dyes into bioconjugable and water-soluble derivatives are described. The combined use of an original sulfonated terminal alkyne and a metal-mediated reaction, namely the copper-catalysed Huisgen 1,3-dipolar cycloaddition ("click" reaction) or the Sonogashira cross-coupling, is the cornerstone of these novel post-synthetic sulfonation approaches.